The infection of a host by an infectious pathogen is a complex event comprising a series of coordinated events in which the pathogen attempts to evade both the host's innate and adaptive immune systems. In their attempts to replicate and survive, the invading pathogens cause damage to the host. The destruction generally takes the form of cell death, either from pathogen entry into the cell or from endo/exo-toxins produced by the pathogen, as well as the induction of host cellular responses that have the ability to cause further tissue damage, scarring or hypersensitivity. Although between 1938 and 1952, the decline in infectious disease-related mortalities decreased 8.2% per year (Armstrong, G L et al., JAMA 1999, 281:61-6), the death rates due to infectious disease have been increasing since the 1980s. A study performed at the National Center for Infectious Diseases demonstrated that the death rate due to infectious diseases as the underlying cause of death increased 58% from 1980 to 1992. In 1992, 64 of every 1,000 deaths were attributable to infectious disease (Pinner, R W et al., JAMA 1996, 275:3).
The mechanisms of infectious diseases comprise two distinct but interconnected aspects: (1) the various organisms attempting to invade a host, each different class of infectious agents having distinct means by which they attempt to evade the host's immune system, and (2) the host's immune response to the invading pathogen. Humans have warded off infectious diseases in both of these aspects, in the first aspect, by preventing the access of infectious agents (e.g. by sanitary habits) and in the second aspect, by assisting and boosting the immune system by, for example, vaccination.
The Fight Against Infectious Disease
Immunization programs in the effort to control infectious diseases, such as small-pox, polio, measles, mumps, rubella, influenza caused by Haemophilus influenza (flu), pertussis, tetanus, and diphtheria, used centuries old technology to safely create an immune response in a host prior to pathogenic infection by the live organism. These vaccination protocols called for the introduction to the host of an inactive or very weak form of the actual pathogen, and in so doing an active immune response was created.
To make vaccination more effective, advances in eliciting stronger immune responses have been made with the development of antigen/epitope non-specific treatments that boost immune activity, such as the adjuvant alum (see Vaccine Adjuvants and Delivery Systems, edited by Manmohan Singh 2007 Wiley & Sons ISBN: 978-0-471-73907-4, incorporated by reference herein, for an extensive review of vaccine adjuvants), as well as development of immunogens based on the understanding of the genetic basis of these pathogens (GenBank, a database managed by the National Center for Biotechnology Information, now has over 85 billion base pairs in its database. Searching based on pathogen is widely used (http://www.ncbi.nlm.nih.gov/Genbank/). The latter has opened up the possibility of utilizing discrete sequences of proteins derived from the pathogen as immunizing agents in the context of cell-mediated (T cell via MHC class I and/or II), but not humoral (Bcell/antibody-mediated immunity). These peptide-based vaccines are specific to epitopes (also called antigenic determinants) that are the precise moieties within antigenic materials to interact with immune system components, intended to boost immune reactivity, and are administered using methods designed to excite immune function. One major limitation of the epitope/peptide-based approach is the variability with which the human MHC class I and II receptors bind the peptides.
While improvements to the techniques have been made in the form of differing types of inactivation of pathogen or in the use of adjuvants to enhance immunogenicity, there remain infectious diseases that are generally refractory to traditional vaccine therapies. In the context of influenza vaccines specific for a particular strain of the virus, clinical efficacy rates range near 40% when there is not a match with the circulating strain (Ben-Yedidia, T and Arnon, R, Expert Rev. Vaccine 2007, 6:939-948).
A need remains in the development of vaccines that can handle the infectious agents such as human immunodeficiency virus (HIV), cytomegalovirus, and severe acute respiratory syndrome coronavirus, as well as bacteria such as Pseudomonas aeruginosa, Neisseria gonorrhea, or Mycobacterium tuberculosis or parasitic diseases such as malaria or hookworm disease. In the context of influenza, the current licensed vaccines are produced in eggs, and make use of half-century old technology (Ben-Yedidia 2007, above).
These infectious agents, bacteria, and parasitic diseases are harder to treat using the inactive pathogen vaccine approach because of the organism's ability to evade host detection. The HIV or the flu virus has the ability to alter its immune profile multiple times in the amount of time less than a calendar year, progressively marginalizing the effectiveness of immunity gained by previous infection and/or even the most recently created vaccine.
In particular, flu viruses, which can easily spread widely and ubiquitously throughout the world, have a large adverse effect on the human population, affecting 10-20% of the total world-wide population (Ben-Yedidia 2007, above). Flu viruses are categorized into three types, A, B, and C. Influenza C rarely infects humans, but influenza A viruses infect various species and B viruses are specific to humans. A and B can be highly virulent. The human immune system attacks flu viruses by targeting their surface proteins hemagglutinin (HA) and neuraminidase (NA). As such, subtypes of flu viruses are categorized as combination of HA type and NA type. HA is a tri-valent viral glycoprotein with two subunits H1 and H2. The virus uses the H1 portion to gain entry to host cells by binding to α2-6 linked sialosides (human), or 2-3 linked sialosides (avian) (Stevens, J et al., J. Mol. Biol. 2008, 381:1382-1394). The secondary and tertiary structures of HA have been solved (Wilson, I A et al., Nature 1981, 289:366-73). Currently there are 15 known subtypes of HA and 9 known subtypes of NA.
The difficulty for the human body to effectively counter influenza infection lies in the fact that the HA protein mutates quickly, allowing the viruses to evade the host organism's immune system. The average rate of amino acid substitutions is 3.6 per year (Smith, D J et al., Science 2004, 305:371-6). Once a mutation occurs in a given epitope region of influenza HA, a second mutation does not immediately occur in that epitope region resulting in a linear profile of temporal appearance of mutation sequence clusters in HA (Plotkin, J B et al., Proc. Nat. Acad. Sci. USA 2002, 99:6263-68). The H1 subunit of HA has been shown to have five main antigenic epitopes. In these regions the amount of mutation is significantly higher than in other regions of the protein.
It is important to understand the metes and bounds of the immunity induced in a host by immunization with a single strain of influenza; how said host responds to another strain dictates the selection of the next vaccine strain. The mutations in the influenza HA protein are subdivided into two groups, antigenic drift and antigenic shift. Antigenic drift is a phenomenon where the protein sequence of HA changes, resulting in immune evasion and improved ability of the virus to enter and replicate in a host cell. Drift measurement metrics have evolved and include the Hamming and Miyata metrics (Miyata, T et al., J. Mol. Evol. 1979, 12:219-36; Henikoff, S et al., Proc. Nat. Acad. Sci. USA 1992, 89:10915-19). Antigenic drift causes the seasonal flu surge that is usually not lethal to otherwise healthy people with normal immune system, but it still affects 10-20% of the world's human population, with up to 5 million cases of serious illness and half million deaths, according to the World Health Organization (WHO). Using the Hamming metric, investigators have suggested that no viral cluster (drifted sequences) has members that last longer than seven years, due to new dominant clusters that replace one another on average every 2-5 years. Other investigators have characterized the inter-pandemic evolution of influenza as intervals of immunologically neutral sequence evolution without significant antigenic alteration. During this time of stasis, there is a slow extinction of coexisting virus lineages that is contrasted with rapid and dramatic excess of amino acid replacements in the variable, or epitopic regions of HA, resulting in the ascension of the new dominant strain at the expense of the earlier variants (Wolf Y I et al., Biology Direct 2006, 34:1-19). These investigators determined that in periods of slow extinction (drift), the ratio of the number of amino acid replacements in the variable, or epitopic regions to the non-variable regions was 9:8, whereas periods of rapid extinction (shift) saw a ratio of 23:1 (Wolf, 2006, above). Thus one means to define a cluster of variants is those currently existing strains that have a ratio smaller than 9:8 of amino acid changes between epitopic and non-epitopic regions.
Antigenic shift, on the other hand, is a major change, requiring four or more amino acid changes across two or more antigenic sites (Plotkin, 2002 above). Such antigenic shift causes an increase in pathology and mortality in humans because of their immunological naiveté to the new strain. In the past, the fast expansions of Spanish flu or Hong Kong flu were deadly. Recently a threat of the H5N1 strain, the “bird flu” more fully described below, turning into a human pandemic strain is emerging.
The modern world has combated influenza epidemics by attempting to predict the next prevalent viral subtype and vaccinating people against it. In 1952, the WHO established the Influenza Program to assist fight against influenza epidemic, and coordinates a network of over 100 influenza surveillance centers all over the world. The centers characterize the antigenic properties of the isolated viruses using a test known as the hemagglutination inhibition (HI) assay, which measures the ability of the virus to agglutinate red blood cells, and the capability of antisera against related strains to inhibit such agglutination. The results of the HI assay is measured in units called antigenic distances, each of which unit corresponding to a twofold dilution of anti-sera in the assay (Smith, 2004, above). Thus a subsequent means to identify a cluster of variants is those currently existing strains having less than a twofold dilution of anti-sera in the HI assay.
In the United States, the Vaccines and Related Biological Products Advisory Committee of the Division of Viral Products (“the Vaccines Committee”) at the Center for Biologics Evaluation and Research (CBER) of the Food and Drug Administration defines the most relevant strains for the production of vaccines using the HI assay. When the antigenic distance is greater than two, the Vaccines Committee will update the variant for the upcoming season (Smith, 2004, above). For the Northern Hemisphere's 2008-9 season, for example, the Vaccines Committee recommended H1N1 A/Brisbane/59/2007 and H3N2 A/Brisbane/10/2007 for Influenza A and B/Florida/4/2006 for Influenza B for the seasonal production of licensed vaccines (inactivated or attenuated viruses).
The choice of the Vaccines Committee of which is the appropriate strain for the upcoming season's vaccine production and distribution is based in large part on antigenicity. Quantitative measurements of antigenic data include numerical taxonomy or its equivalent (Papaud, A, Poisson A, J Mar Res 1986, 44:385; Mecking S, Warner J, Geophys Res 1999, 104:11087), the method of Lapedes and Farber (Wallace D W R, Ocean Circulation and Climate Academic Press San Diego, Calif. 2001 pp 489-521) which uses ordinal multidimensional scaling in the interpretation of binding data so that an antigenic map, or the distance between an antigen and antiserum can be visualized. A refinement of the Lapedes and Farber method (Smith D J, 2004, above; Sabine C L et al., Global Biogeochem Cycles 16:1083, 2002) positions/overlays the antigens and the antisera on a map.
An alternate approach of defining clusters is differentiation by the organization of genetic (DNA) data into phylogenetic trees. The international surveillance programs are responsible for the majority of the sequencing performed in order to identify serologically novel influenza strains (Layne S P, Emerg. Infect. Dis. 12:562-68, 2006). Investigators have reported that the meaningful antigenic changes that occurred in an H3N2 subtype influenza virus during the period between 1983 ad 1997 were found predominantly in the internal and not external branches of the tree (Bush, R M et al. Mol Biol Evol 1999, 16:1457-65). Thus a further subsequent means to identify a cluster of variants is the branching organization of those currently existing strains obtained using genetic data.
There are various types of vaccines available for human use: DNA-based, cellular, live pathogen, attenuated or inactivated pathogen-based, recombinant protein and peptides. There are currently five inactivated seasonal vaccines for human use (Fluzone®, Fluvirin®, Fluarix®, FluLaval®, and Afluria®), and one live attenuated (FluMist®). Peptide based vaccines can be either recombinant or synthetic. Since 1993 there have been over 1,000 peptide based vaccines in pre-clinical research, over 100 in Phase I human clinical trials, a small few in Phase II, and none passing Phase III (Hans, D et al., Medicinal Chemistry 2006, 2:627-46).
The candidate viral subtypes for annual flu vaccine update do not include the mainly avian infective H5N1, which have historically infected poultry. However, investigators at Scripps Research Institute and others have identified the Egret/Egypt/1162/NAMRU-3/06 as a variant having likely human infective potential (Stevens, 2008, above). In fact, reports of the H5N1 viruses infecting humans are increasing. Because of the lack of exposure to related sub-strains, an H5N1 infection in a human often has severe effects. The recently emerging ability of the virus to infect humans is thought to be attributable to the conversion from a SAα2-3Gal to SAα2-6Gal receptor recognition by the virus. Investigators have found that H5N1 virus that infected humans is capable of recognizing both types of receptors (Yamada, S et al., Nature 2006, 444:378-82).
Although helpful to an extent in preventing the disease, the success of the WHO's Influenza Program is limited for several reasons. One such reason is the limited number of surveillance locations and a small sampling size compared to the occurrence of influenza infection (in 2004-2005, about 6000 samples, or 1 sample per 100,000 cases). Another drawback is the time it takes to prepare vaccines once such candidate antigenic subtypes have been selected (Layne, Emerging Infectious Disease 2004, 12(4): 562-568). Added to the difficulties is the above-mentioned antigenic drift and shift. New strains may appear at any time.
Another complicating phenomenon is broadening of the immune response via the process of epitope spreading, where a host's immune system over time changes target of the immune attack to nearby epitopes (N. Suciu-Foca et al., Immunol. Rev. 1998, 164:241). Thus, the inability to modulate the relevant antigenic determinants over time renders simple vaccines ineffective in a short time.
Therefore, a new method for developing and preparing vaccines that are effective under these circumstances are needed.
Strategy for Creating Active Peptides
In recent years, vaccines based on synthetic peptide epitopes rather than whole antigenic molecules have been explored and developed, in the hope of more refined targeting and ease of achieving consistent quality, as well as the safety of being non-infectious. The results are mixed. As a practical matter, it is time- and resource-intensive to identify one peptide or a set of a limited number of peptides that would be effective as a vaccine. In addition, because of the infectious agents' immune evasion strategies briefly described above, peptide epitope-based vaccines have not overcome the shortcomings of traditional vaccines. Investigators have attempted to make use of conserved epitopes within the virus. These regions do not mutate, and thus represent an approach for the generation of broad-spectrum, peptide-based vaccines (Ben-Yedidia, 2007, above). These approaches using short peptides; however, the invoked immunity is T cell-based, requires adjuvant or carrier proteins (flagella) and lacks the proven pathogen fighting ability of preexisting antibodies (Zinkernagel, R M, Nobel Lecture Dec. 8, 1996).
The development and exploitation of combinatorial chemistry (CC) has propelled drug discovery. Drug discovery can be generalized into two major steps, lead generation and lead optimization. Oftentimes, a lead compound is identified that has some of the desired characteristics of a commercially viable therapeutic, but has shortcomings such as a low specific activity, toxicity, instability, etc. Thus, once a lead is identified, practitioners attempt to optimize the lead compound by testing other related compounds with similar structures. CC allows practitioners to create and quickly screen a library made of a vast number of candidates, to identify those with a specific activity against a target of interest.
For peptide based drugs, the goal is to define a single, or a limited set of peptides which demonstrate a particular activity. The art of CC as applied to the synthesis of peptide libraries, too, has advanced, producing highly reliable and pure mixtures of peptides of great diversity. The process of identifying the single or limited set of peptides that were responsible for the observed activity from such diverse libraries, called deconvolution, is schematically represented in FIG. 1A.
Drug discovery based on CC are powerful, but deconvolution can be difficult and time- and cost-intensive. Thus, to simplify deconvolution and make the process more efficient, practitioners have spent great efforts to create suitable synthesis methods. Examples of the resulting evolution of subtypes of combinatorial methods include: multiple synthesis, iterative synthesis, positional scanning, and one-compound-one-bead post assay identification design. They are briefly described below to show the state of the art and how peptide epitopes were successfully identified.
“Multiple synthesis” provides for any method whereby distinct compounds are synthesized simultaneously to create a library of isolated compounds. The identity of these compounds would be known from the rules of the synthesis. An example is found in H. M. Greysen et al., Proc. Nat. Acad. Sci. USA, 1984, 81:3998, where the investigators identified GDLQVL (SEQ ID NO: 20), out of 108 overlapping peptides, as the epitope recognized by an antibody raised against VPI protein of foot-and-mouth disease virus.
“Iterative synthesis/screening” involves methods of peptide synthesis which facilitates the determination of the peptide sequences as deconvolution is performed. This is done by synthesizing pools of peptides, each pool consisting of peptides with the two amino terminal residues defined and known to the investigators. These peptides can be shown as O1O2XXXX, wherein O1 and O2 are the defined residues and X is randomly selected. When a pool that contains a peptide that binds to a target is identified, the two amino terminal residues are known. The next step was to do the same for position three, by synthesizing peptides that can be shown as F F2O3XXX, wherein 03 is a fixed residue. Subsequent residues are determined in a similar manner. An example of iterative synthesis can be seen in R. A. Houghten et al., Nature 1991, 354:84-86, wherein the investigators honed in on the sequence DVPDYA (SEQ ID NO: 21) as the core epitope after identifying YPYDVPDYASLRS (SEQ ID NO: 22) using an ELISA type assay format.
“Positional scanning” is a synthesis method producing complex mixtures of peptides that allows for the determination of the activity of each individual peptide. Based on the screening results, the derived peptide can then be separately synthesized for optimization. As seen in C. Pinilla et al., Biochem J. 1994, 301:847-853, positional scanning libraries were used to identify decapeptides which bound the same YPYDVPDYASLRS-binding antibody (SEQ ID NO: 22). In this case ten different libraries each containing 20 pools with a defined amino acid at each of the ten positions in the peptide. Fifteen peptides were identified.
Each of the above methods were also employed to identify enzyme substrates (J. H. Till et al., J. Biol. Chem. 1994, 269:7423-7428, J. Wu et al, Biochemistry 1994, 33:14825-14833, W. Tegge et al., Biochemistry 1995, 34:10569-10577) or enzyme inhibitors (M. Bastos et al., Proc. Nat. Acad. Sci. USA 1995, 92:6738-6742, Meldal et al., Proc. Nat. Acad. Sci. USA 1994, 91:3314-3318, R. A. Owens et al., Biomed Biophys. Res. Commun. 1994, 181:402-408, J. Eichler et al., Pept. Res. 1994, 7:300-7).
As powerful and clear-cut the identification of a specific peptide may be, as a therapeutic, such identified peptide may only be a lead peptide that is not itself useful. The identified peptide epitope may be ignored by the immune system if it resembles a self protein or possibly exacerbate the very condition that the therapy aims to relieve. However, by designing peptides of similar sequence, one may create, based on such peptide, epitope reactive analogs that would act as modifiers of the immune responses and/or as excitors of an immunogenicity response.
One such approach is creation of altered peptide ligands (APL). This approach is schematically represented in FIG. 1B. An APL is defined as an analog peptide which contains a small, number of amino acid changes from a starting sequence such as that of a native immunogenic peptide ligand. While recognition of the native response may induce an antagonist-like reaction, an APL might also induce a partial agonist response, or induce a state of anergy in the reactive T cell population. In discussing APL in the context of allograft rejection therapy, Fairchild et al., Curr. Topics Peptide Protein Res. 2004, 6:237-44, note that an APL acting as an antagonist for one TCR, may become an agonist for another, complicating the rational design of an APL. Compounding the obstacle of the development of APL is the difficulty in translating a response developed in an animal system into human.
An illustrative example is an APL based on an epitope of myelin basic protein, MBP83-99 (ENPVVHEFKNIVTPRTP (SEQ ID NO: 23)), which is reported to be a target of autoimmune response causing multiple sclerosis. APLs were made by replacing the bold and underlined amino acid residues “E”, “N”, “E” and “K,” resulting in identification of a single peptide having the amino acid residue sequence AKPVVHLFANIVTPRTP (SEQ ID NO: 24) that appeared to have the desired activity of neutralizing antibodies against MBP83-99, Kim et al. Clinical Immunology, 2002, 104:105-114. The peptide was placed into limited human trials, which reportedly resulted in the long term immune reactivity against the peptide, but the treatment has been deemed clinically ineffective by evaluation using MRI. Thus an APL, once identified, can be used as a therapeutic agent; however, its effectiveness may be limited in terms of clinical efficacy.
Evasive Targets
In the context of developing vaccines in the prevention of infectious disease, the invading pathogens have acquired by natural selection the ability to quickly create variability in the relevant immunogenic epitope. Examples of viral mutants generating escape variants that avoid immune detection have been reported for Hepatitis B, influenza, and HIV (Tabor, E, Journal of Med. Virol., 2006, 78:S43-S47; Daniels, R S et al., EMBO Journal 1987, 6:1459-65; D'Costa, S et al., AIDS Res Hum Retroviruses 2001, 17:1205-9). This variation progressively dampens the utility of single-epitope vaccines.
Thus, investigators have attempted to overcome this limitation by introducing variance by creating a mixture of immunity-inducing epitopes to be used as vaccines. Furthermore, it has previously been shown that mixtures of related peptides may be therapeutically more effective than a single peptide. Lustgarten et al., J. Immunol. 2006, 176: 1796-1805; Quandt et al., Molec. Immunol. 2003, 40: 1075-1087. The effectiveness of a peptide mixture as opposed to a single peptide is the likelihood of interaction with the broadening of the relevant epitopes via evolution-induced variation. Therefore, to increase and maintain the longer-term effectiveness, these previous treatment modalities have been modified. For example, a therapeutic composition based on an APL may include multiple peptides created by the APL method in combination with the original peptide, or other APLs. Fairchild et al., Curr. Topics Peptide & Protein Res. 2004, 6:237-44. Each APL would have a defined sequence, but the composition may be a mixture of APLs with more than one sequence. A reverse example involving conceptually similar altered peptide ligands involves an inventor's attempt to reduce the amount of variation created by pathogens to avoid immune recognition (viral alteration of immunogenic epitopes over time, e.g., the creation of altered peptide ligands), by using the very changes created by the pathogen in an epitope sequence to create a limited diversity pool of peptides potentially useful in vaccinations (U.S. Pat. No. 7,118,874). In this invention, distinct hyper-Variable region sequences from HCV or influenza virus were compared. The amino acid changes having a frequency greater than 12% were incorporated into a mixture of between 2 and 64 different peptides.
However, a simple mixture of several soluble peptide epitopes faced certain manufacturing issue of soluble peptide mixtures, such as difficulties in delivering a consistent ratio and quantity of each of the peptides in the mixture.
Peptide dendrimers were conceived to overcome some of these shortcomings of soluble peptide mixtures, and also by providing other perceived advantages. This approach is schematically represented in FIG. 1C. The design of dendrimers intends to mimic two traits of naturally occurring biological structures: a globular structure and polyvalency to amplify the ligand:substrate interaction. Beyond these commonalities, dendrimers are chemically and structurally diverse, with reports of a wide range of sizes, from 2 kDa to greater than 100 kDa. Dendrimers are described in two comprehensive reviews (P. Niederhafner et al., J. Peptide Sci. 2005 December; 11(12):757-788; K. Sadler and J. P. Tam, Rev. Mol. Biotechnol. 2002, 90:195-229), and their applications in additional literature (D. Zanini and R. Roy, J. Org. Chem. 1998, 63:3468-3491; J. Haensler and F. C. Szoka, Bioconjug Chem. 1993, 4:372-379; Tam, James P et al., J. Exp. Med. 1990, 171:299-306).
Despite attempts and a certain amount of success in utilizing dendrimers and some improvements in synthesis and purification strategies, a high cost of manufacturing and the subsequent analytical development precludes this technology from being further currently developed commercially.
All of the above strategies, while recognizing the advantage of variations in the therapeutic peptide compositions, derive from the concept that there is one or more defined peptide sequences evoking a defined immunological response. These strategies have attempted to multiply and diversify modulatory peptides via the introduction of defined, single changes performed one at a time.
An entirely different approach which has evolved alongside the defined sequence peptide immunotherapy approach is the use of random epitope polymers. Random sequence polymers (RSP) can be described as a random order mixture of amino acid copolymers comprising two or more amino acid residues in various ratios, forming copolymers by random sequence bonding, preferably through peptide bonds, of these amino acid residues, which mixture is useful for invoking or attenuating certain immunological reactions when administered to a mammal. Because of the extensive diversity of the sequence mixture, a large number of therapeutically effective peptide sequences are likely included in the mixture. In addition, because of the additional peptides which may at any given time not be therapeutically effective, but may emerge as effective as the epitope shifting and spreading occurs, the therapeutic composition may remain effective over a time of dosing regimen. This approach is schematically represented in FIG. 1D.
Copolymer-1 (also known as Copaxone®, glatiramer acetate, COP-1, or YEAK random copolymer) is an FDA approved, commercially available therapeutic used for the treatment of multiple sclerosis comprising random copolymers of Y, E, A, and K. Random copolymers are described in International PCT Publication Nos. WO 00/05250, WO 00/05249; WO 02/59143, WO 0027417, WO 96/32119, WO/2005/085323, in U.S. Patent Publication Nos. 2004/003888, 2002/005546, 2003/0004099, 2003/0064915 and 2002/0037848, in U.S. Pat. Nos. 6,514,938, 5,800,808 and 5,858,964. It is one of the few therapeutics for multiple sclerosis that continues to be effective over time.
Attempts continue to build on the success of COP-1 by creating other RSPs and related peptide compositions. WO/2005/074579 (the '579 publication) describes complex peptide mixtures of various amino acid composition. The disclosure also contains diversity-constraining mechanisms of defining amino acids at certain positions rather than being chosen by the random nature of the synthesis rules. Another such attempt is the work originated by Strominger et al. (WO/2003/029276) and developed further by Rasmussen et al. (US 2006/0194725) wherein they created RSP consisting of the amino acids Y, F, A, and K with different amino acid ratios and a shorter average length than COP-1 Alanine content was increased based on Maurer (Pinchuck and Maurer, J. Exp Med 122(4), 673-9, 1965), which described how an EAK polymer with higher alanine content (10-60 mole percent) produced “better antigens”; Rasmussen et al. in fact demonstrated that a YFAK input ratio of 1:1:1:1 was not effective in eliciting a recall response as compared to a YFAK preparation with an input ratio of 1:1:10:6.
WO/2005/032482 (the '482 publication) describes another approach, which is to build degenerate peptide sequences based on motifs, exemplified by [EYYK]. The motifs are used as is, or can be altered by amino acid substitutions (defined on page 10-11 of the '482 publication). A significant difference from COP-1 is that it lacks alanine. Much of the invention hinges on the presence of a D-amino acid at the amino terminal of the motif.
Tracing back steps to the defined peptide search, there have also been attempts to identify the active peptide(s) within the RSP mixture. The drawback of this technology lies in the very nature of the attempt to determine discrete substitutes for the randomness that COP-1 encompasses.
Effective as the random sequence polymer approach may be, even the improvements have not resolved the drawback and limitation of COP-1, which is, for example, the undefined nature of what is effective in each motif and the possibility of containing a large proportion of truly inactive peptides, lowering the concentration of the active components, or worse, adversely stimulating the immune system. Additionally, these compounds are difficult to manufacture and to obtain consistency from lot-to-lot.
All these approaches have not overcome the shortcomings of previously existing systems, and need remains for a composition and a method to create such composition that would serve effectively as a vaccine by eliciting beneficial immune responses consistently and over time toward pathogens, for which existing vaccine compositions have failed to be effective.